International Journal of

Molecular Sciences

Review
Deciphering SARS-CoV-2 Virologic and
Immunologic Features
Grégorie Lebeau 1,2, *,† , Damien Vagner 1,3,† , Étienne Frumence 1,2 , Franck Ah-Pine 4 ,
Xavier Guillot 1,5 , Estelle Nobécourt 6,7 , Loïc Raffray 8 and Philippe Gasque 1,2
1 Unité de Recherche Études Pharmaco-Immunologiques, Centre Hospitalier Universitaire La Réunion Site
Félix Guyon, CS11021, 97400 Saint Denis de La Réunion, France; damien.vag@gmail.com (D.V.);
etienne.frum@gmail.com (É.F.); xavier.guillot@chu-reunion.fr (X.G.); Philippe.gasque@chu-reunion.fr (P.G.)
2 Laboratoire de Biologie, Secteur Laboratoire d’immunologie Clinique et Expérimentale de la Zone de l’océan
Indien (LICE-OI), Centre Hospitalier Universitaire La Réunion Site Félix Guyon, CS11021,
97400 Saint Denis de La Réunion, France
3 Unité Mixte de Recherche Processus Infectieux en Milieu Insulaire Tropical (PIMIT), Université de La
Réunion, INSERM UMR 1187, CNRS 9192, IRD 249, Platform CYROI, 2 rue Maxime Rivière,
97491 Sainte Clotilde, La Réunion, France
4 Service d’anatomo-Pathologie, Centre Hospitalier Universitaire Sud Réunion, 97410 Saint Pierre, France;
franck.ahpine@gmail.com
5 Service de Rhumatologie, Centre Hospitalier Universitaire La Réunion Site Félix Guyon, CS11021,
97400 Saint Denis de La Réunion, France
6 Service d’endocrinologie Diabétologie, Centre Hospitalier Universitaire Sud Réunion,
97410 Saint Pierre, France; estelle.nobecourt@chu-reunion.fr
7 Université de Formation et de Recherche Santé, Université de la Réunion, 97400 Saint-Denis, France
8 Service de Médecine Interne, Centre Hospitalier Universitaire La Réunion Site Félix Guyon, CS11021,
97400 Saint Denis de La Réunion, France; loic.raffray@chu-reunion.fr
* Correspondence: greg.lebeau@live.fr; Tel.: +262-692-55-05-83
† Both authors contributed equally to this work.




Received: 19 June 2020; Accepted: 14 August 2020; Published: 18 August 2020


Abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and its associated pathology,
COVID-19, have been of particular concerns these last months due to the worldwide burden they
represent. The number of cases requiring intensive care being the critical point in this epidemic,
a better understanding of the pathophysiology leading to these severe cases is urgently needed.
Tissue lesions can be caused by the pathogen or can be driven by an overwhelmed immune response.
Focusing on SARS-CoV-2, we and others have observed that this virus can trigger indeed an immune
response that can be dysregulated in severe patients and leading to further injury to multiple
organs. The purpose of the review is to bring to light the current knowledge about SARS-CoV-2
virologic and immunologic features. Thus, we address virus biology, life cycle, tropism for many
organs and how ultimately it will affect several host biological and physiological functions, notably
the immune response. Given that therapeutic avenues are now highly warranted, we also discuss
the immunotherapies available to manage the infection and the clinical outcomes.

Keywords: SARS-CoV-2; COVID-19; virus biology; immunopathology; immunotherapy

1. Introduction
A series of coronaviruses crossed the species barrier, with the severe acute respiratory syndrome
coronavirus (SARS-CoV) in 2003 and the Middle East respiratory syndrome coronavirus (MERS-CoV)
in 2012, and they caused major outbreaks of lethal pneumonia. Now, a new coronavirus emerged in

Int. J. Mol. Sci. 2020, 21, 5932; doi:10.3390/ijms21165932 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 5932 2 of 40

December 2019 in Hubei province and was named the severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) virus. The disease caused by this virus, called COVID-19 has spread worldwide causing
as early as August 2020 more than 18 million cases and more than 700,000 deaths [1].
SARS-CoV-2 is an enveloped virus with a positive RNA genome belonging to the beta genus of
the Coronaviridae family [2].
The clinical features of COVID-19 range from asymptomatic or mild disease to critically
life-threatening ill patients conditions. The most prevalent symptoms are fever, shortness of breath,
dyspnea, cough and fatigue [3] and are commonly associated with diarrhea, headache, anosmia,
lymphopenia or liver injury. An increasing number of less common manifestations are being described
like acute kidney injury, vasculitis or myocarditis [4]. The most common comorbidities in critically
ill patients are age, hypertension, diabetes, obesity, cardiovascular disease and respiratory system
disease [3].
Viral and host interaction plays a key role in the pathogenesis of the disease. With the pandemic
diffusion, better knowledge of the viral life cycle, cell tropism and host immune response is needed
to find treatment against the disease. Here, we review the current understanding of the viral cycle,
immunopathology as well as the current progress of immunotherapy.

2. Viral Description

2.1. Unravelling SARS-CoV-2 Genome and Viral Protein Features

SARS-CoV-2 (previously named new coronavirus 2019, nCoV-2019) is a single-stranded positive
RNA virus, classified among the family Coronaviridae, within the Nidovirales order [2]. More
precisely, SARS-CoV-2 belongs to the subgenus Sarbecovirus, within the Betacoronavirus genera and
the subfamily Orthocoronavirinae [5,6]. Like other coronaviruses, SARS-CoV-2 was shown to possess
a long genome, near to 30 kb, which showed 80% similarity with SARS-CoV, 50% with MERS-CoV
and more identity with bat related coronaviruses (88% with bat-SL-CoVZC45 and bat-SL-CoVZXC21,
93% with RaTGT13) [6–9]. The first 2/3 parts of the genome encode the replicase–transcriptase
complex, and the last third of the genome the four structural proteins [5,8,9]. Among all viral proteins,
the replicase–transcriptase complex is the only one directly translated from the genome. ORF1a and
ORF1b encode for two polyproteins (pp1a and pp1b) autoproteolytically processed to give the 16
non-structural proteins [5,10]. Whereas the structural proteins spike (S), envelope (E), membrane (M),
nucleocapsid (N) and other accessory ones are expressed from subgenomic mRNAs [5,10].
The SARS-CoV-2 virion has a diameter of approximately 125 nm. The virion surface mainly bears
the M protein and is poor in the E protein, however this last one is crucial for intracellular membrane
assembly [5,11–13]. Moreover, the virion surface presents the spike protein (S), a highly glycosylated
petal-shaped protein, composed of two functional domains S1 and S2 responsible for binding to
the host cell receptor and membrane fusion respectively. The long genome is associated with helically
symmetric N protein to form a nucleoprotein (NP), enclosed in the virion envelope. As SARS-CoV-2 has
been recently identified, little is known about a large part of its proteins (structural and non-structural).
However, growing efforts are made for this purpose. The SARS-CoV-2 spike protein is one of the most
described, as it is the one that gives its infectivity to the virus. Spikeless coronaviruses exert poor
infectivity [5]. Hence, this protein is essential for viral entry, as it binds to Angiotensin Converting
Enzyme II—ACE2 as an entry receptor on the host cell, through its receptor-binding domain (RBD)
located in the S1 subunit [14]. This system of entry is shared with SARS-CoV [15–18]. Additionally, this
S protein is one of the major antigens [5], against whom is directed the neutralizing antibodies [14,19].
Notably a highly conserved epitope has been identified [20], which may explain cross-neutralization
by SARS-CoV S-specific antibodies [14,17,19].
Of note, near-atomic structure of SARS-CoV-2 nsp12-nsp7-nsp8 complex (respectively catalytic
subunit and two cofactors), representing the core polymerase complex, has been described and highly
resembles SARS-CoV. Additionally, this gives insights about a better adaptation to the human host
Int. J. Mol. Sci. 2020, 21, 5932 3 of 40

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SARS-CoV 21, x FOR PEER
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[21], 3 of 40
maybe explaining the increased ribonucleic acid (RNA) production
compared to SARS-CoV [22]. Finally, the SARS-CoV-2 main protease (Mpro) X-ray structure has also
human host than SARS-CoV polymerase [21], maybe explaining the increased ribonucleic acid
been reported [23]. Among the viral proteins that we presented, some are potential targets for novel
(RNA) production compared to SARS-CoV [22]. Finally, the SARS-CoV-2 main protease (Mpro)
drugX-ray
development
structure has or drug repurposing,
also been withAmong
reported [23]. alreadytheestablished lead
viral proteins compounds,
that notably
we presented, due to
some are
computational
potential targets for novel drug development or drug repurposing, with already established leadneed
methods [18,23,24]. The antiviral activity and clinical benefits of these compounds
to becompounds,
investigated. notably due to computational methods [18,23,24]. The antiviral activity and clinical
benefits of these compounds need to be investigated.
2.2. Inside the SARS-CoV-2V Life Cycle
2.2. Inside the SARS-CoV-2V Life Cycle
2.2.1. Entry and Fusion
2.2.1. Entry and is
SARS-CoV-2 Fusion
an airborne virus, meaning that it can be transmitted through droplets or
contacts.SARS-CoV-2
Subsequently, is an
theairborne virus, in
virus enters meaning thatDifferent
its target. it can beroads transmitted
of entrythrough droplets
have been or
identified
contacts.
(Figure Subsequently,
1A). First, the most the virus enters
described, in its target. Different
as aforementioned roads of ACE2
passes through entry have
due to been
theidentified
RBD of the S
(Figure
protein 1A). First,
[15–17]. the most
This entry alsodescribed, as aforementioned
implies the cleavage of the passes through
S protein, ACE2
by the due to the RBD
transmembrane of
protease
the S protein [15–17]. This entry also implies the cleavage of the S protein, by
serine 2 (TMPRSS2), which allows it to release the fusion peptide contained into the S2 domain and the transmembrane
protease
largely serine
increases 2 (TMPRSS2),
SARS-CoV-2 which
entry, allowstoitwhat
similarly to release the fusion
was shown peptide contained
for SARS-CoV [14–16].into the S2
Alternatively,
domain and largely increases SARS-CoV-2 entry, similarly to what was shown for SARS-CoV [14–
a cleavage by the cathepsin L during endocytosis is also possible. Interestingly, alike the classical
16]. Alternatively, a cleavage by the cathepsin L during endocytosis is also possible. Interestingly,
cleavage site known in the SARS-CoV S protein, SARS-CoV-2 S presents a furin-like cleavage site, often
alike the classical cleavage site known in the SARS-CoV S protein, SARS-CoV-2 S presents a
observed in highly virulent influenza viruses. This may permit cleavage in the Golgi apparatus during
furin-like cleavage site, often observed in highly virulent influenza viruses. This may permit
biosynthesis
cleavage in (the
theglobal
Golgistructure
apparatusstill maintained
during by non-covalent
biosynthesis (the global bonds)
structureand stillextend tropism
maintained by and
transmissibility,
non-covalent bonds) and extend tropism and transmissibility, due to the near-ubiquitous expression of
due to the near-ubiquitous expression of furin-like protease [14]. An abundance
of furin-like
literature protease ACE2/RBD
demonstrated [14]. An abundance
binding, but of literature
recently, a demonstrated
new route ofACE2/RBD
entry has been binding, but via
evoked
CD147recently, a new
(Basigin) route
and of entry
needs to behas been evoked
further explored via[25],
CD147as (Basigin)
these resultsand needs to questionable.
are still be further explored
[25], as these results are still questionable.

Figure
Figure 1. SARS-CoV-2
1. SARS-CoV-2 supposed
supposed lifelife cycle.(A)
cycle. (A)Entry
EntryofofSARS-CoV-2
SARS-CoV-2 in
in target
target cell
cell expressing
expressing ACE2
ACE2 (or
(or another receptor, CD147 have been evoked but need to be confirmed).
another receptor, CD147 have been evoked but need to be confirmed). (B) Uncoating (B) Uncoating and
and releasing
Int. J. Mol. Sci. 2020, 21, 5932 4 of 40

SARS-CoV-2 single stranded positive RNA genome. (C) Translation of replicase–transcriptase complex
directly from RNA genome. (D) RNA genome replication due to a negative template. (E) Nested
production of subgenomic RNA encoding for structural proteins. (F) Translation of viral S, E and M
inserted in endoplasmic reticulum. (G) Nucleocapsid coupled to the genome, forming nucleoprotein,
combine to S, E and M to form a mature virion (H). (I) Exocytosis of SARS-CoV-2.

2.2.2. Release and Replication

The process of entry and fusion lead to virion contents (i.e., nucleocapsid) release inside the targeted
cell (Figure 1B). The next step in the viral cycle is the replicase–transcriptase complex translation
directly from the genomic RNA (Figure 1C), a process requiring ribosomal frameshifting [10,26].
The encoded polyprotein, as said earlier, is processed to give 16 nsp, which assemble to form
the replicase–transcriptase complex. In coronaviruses, this complex exerts the particularity to be
associated with remodeled cellular membranes, only dedicated to welcome viral RNA synthesis [27,28],
representing a mean of immune evasion. Afterwards, negative-sense RNA intermediates are generated
to serve as templates for the synthesis of genomic RNA on the one hand, and subgenomic RNA on
the other hand that will encode structural proteins and several accessory proteins (Figure 1E) [29].

2.2.3. Assembly
After translation, M, S and E proteins are inserted in the endoplasmic reticulum (ER) membrane
(Figure 1F). From this location, they are transported, through a secretion pathway, to the site of
assembly: the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) [10,30]. Assembly
requires a complex cooperation between the M protein and other structural proteins, notably the E
protein, which is essential for assembly [5,10,30,31]. N protein complexes with newly synthesized
genomic RNA in cytoplasm to form NP, which then reaches the ERGIC to which achieves progeny
viral formation by condensation of NP with the envelope components (Figure 1G). Conversely to what
is known for entry and fusion of SARS-CoV-2, few data are available at this time about other steps
of the viral life cycle. Hence experiments are highly awaited to address the direct cytopathic effects
of the SARS-CoV-2 replicative cycle on cells that support infection [32,33]. Of note immune-induced
tissue injuries need to be taken into consideration and these will be discussed largely below.

2.2.4. Budding and Transmission

Finally, the last step of the virus life cycle is virion exocytosis (Figure 1I). Virus budding appears
to be done near the Golgi apparatus, presumably into smooth membranes of ERGIC [28]. This leads to
two phenomena: transmission and viral spreading in other organs. Transmission is mainly described
by person-to-person airborne transmission, via droplets or contact [34–36]. However, other routes
of transmission are supposed (such as orofecal or ocular), as the virus can be found in the digestive
tract [37,38] and ocular surface express ACE2 [39].
Even if data might be extrapolated from what is known from other coronaviruses, going forward
remains crucial to confirm them with a cellular model of infection.

2.3. Tropism and Associated Clinical Manifestations

As we have seen, SARS-CoV-2 is mainly supposed to enter via a principal surface protein ACE2
(and maybe other entry receptors/co-receptors such as CD147) and fuse via TMPRSS2 or furin (Figure 2).
These proteins are expressed on several cell types and tissues, explaining the multiple organ injuries
that can be observed in SARS-CoV-2 infection.
Int. J. Mol. Sci. 2020, 21, 5932 5 of 40
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 40

Figure
Figure 2. The
2. The two
two evokedroutes
evoked routesof
of entry
entry for
for SARS-CoV-2
SARS-CoV-2totodate. Angiotensin-converting-enzyme
date. Angiotensin-converting-enzyme 2
(ACE2), which has been described as an interferon-stimulated gene (ISG), is a route of
2 (ACE2), which has been described as an interferon-stimulated gene (ISG), is a route of entry forentry for
SARS-CoV-2.
SARS-CoV-2. Additionally,
Additionally, CD147
CD147 is evoked
is evoked as aas a potential
potential second
second routeroute of entry.
of entry. BasedBased on a
on a previous
previous study with SARS-CoV, an interaction with Cyclophilin A is possible. The blue background
study with SARS-CoV, an interaction with Cyclophilin A is possible. The blue background corresponds
corresponds to cells expressing ACE2, whereas the red background is representing cells expressing
to cells expressing ACE2, whereas the red background is representing cells expressing CD147. Solid
CD147. Solid arrows correspond to a direct activity involving ACE2, dotted arrows correspond to an
arrows correspond to a direct activity involving ACE2, dotted arrows correspond to an indirect
indirect promoting activity.
promoting activity.
ACE2 is a carboxypeptidase responsible for the conversion of angiotensin II (a potent
ACE2 is a carboxypeptidase responsible for the conversion of angiotensin II (a potent
vasoconstrictor) to angiotensin (1–7; a vasodilator). ACE2 is expressed in the nose, lung, ileum,
vasoconstrictor) to angiotensin (1–7; a vasodilator). ACE2 is expressed in the nose, lung, ileum,
heart, eye, liver, bladder, kidney, pancreas, brain, prostate, testis and placenta [40–42]. Following
heart,
binding of the bladder,
eye, liver, SARS-CoV-2 kidney, pancreas,
S protein, ACE2brain,
activityprostate, testis and
and its surface placenta
expression are [40–42].
decreased. Following
Thus,
binding of the
the loss SARS-CoV-2
of ACE2 expressionS protein,
on the cellACE2 activity
surface mayand its an
create surface expression
unbalance in the are decreased. Thus,
renin–angiotensin
system,
the loss resulting
of ACE2 in reduced
expression on themetabolization
cell surface may of angiotensin II and leaky
create an unbalance pulmonary
in the blood vessels
renin–angiotensin system,
associated with severe acute lung injury through Angiotensin II Type 1a receptor stimulation
resulting in reduced metabolization of angiotensin II and leaky pulmonary blood vessels associated
with(AT1R)
severe[43,44]. Additionally,
acute lung it has been
injury through shown that
Angiotensin the injection
II Type 1a receptorof recombinant
stimulationSARS-CoV S
(AT1R) [43,44].
protein itself
Additionally, in acid
it has been aspiration-induced acute lung
shown that the injection of injury in the mouse
recombinant is sufficient
SARS-CoV to boost
S protein itselflung
in acid
failure [43]. It may
aspiration-induced be that
acute lungsimilar
injuryeffects
in theofmouse
SARS-CoV-2 S protein
is sufficient could lung
to boost be envisaged. It hasIt been
failure [43]. may be
shown that SARS-CoV binding to ACE2 could lead to ACE2 shedding
that similar effects of SARS-CoV-2 S protein could be envisaged. It has been shown that SARS-CoV as a soluble form by
ADAM17, also known as a tumor necrosis factor alpha (TNF-α) converting
binding to ACE2 could lead to ACE2 shedding as a soluble form by ADAM17, also known as a tumor enzyme [45–48].
Inflammatory cytokines such as TNF-α could enhance ACE2 shedding [45,46]. Of further note, it is
necrosis factor alpha (TNF-α) converting enzyme [45–48]. Inflammatory cytokines such as TNF-α
known that AT1R directly upregulates nuclear factor-κB (NF-κB) [49–51] and ADAM17 [52], which
could enhance ACE2 shedding [45,46]. Of further note, it is known that AT1R directly upregulates
in turn can cleave membrane-bound TNF to produce soluble TNF. So, we may have here a vicious
nuclear factor-κB (NF-κB) [49–51] and ADAM17 [52], which in turn can cleave membrane-bound TNF
spiral where ACE2 loss leads to overstimulation of the AT1R axis and subsequent upregulation of
to produce soluble TNF. So, we may have here a vicious spiral where ACE2 loss leads to overstimulation
NF-κB and ADAM17, triggering ACE2 shedding enhancement. TMPRSS2 can also participate in the
of the
processingaxis
AT1R and subsequent
of ACE2 [53]. Solubleupregulation
ACE2 remains of NF-κB and ADAM17,
biologically active andtriggering
competes ACE2 shedding
for S protein
enhancement.
binding. Furthermore, endocytosis of ACE2 after binding to SARS-CoV has been reported remains
TMPRSS2 can also participate in the processing of ACE2 [53]. Soluble ACE2 [54].
Interestingly,
biologically it has
active andbeen reported
competes forthat ACE2 isbinding.
S protein an interferon-stimulated gene in vitro, of
Furthermore, endocytosis suggesting
ACE2 after
that the
binding to cell inflammatory
SARS-CoV response
has been could [54].
reported indirectly enhance SARS-CoV-2
Interestingly, it has beeninfection
reported [55].
that ACE2 is an
interferon-stimulated gene in vitro, suggesting that the cell inflammatory response could indirectly
enhance SARS-CoV-2 infection [55].
Int. J. Mol. Sci. 2020, 21, 5932 6 of 40

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 40

Other receptors have been reported to mediate SARS-CoV infection. DC-SIGN and L-SIGN serve
Other receptors
as alternative receptors for
have been reported
SARS-CoV entryto mediate SARS-CoV
independently of ACE2 infection.
[56–58].DC-SIGN and L-SIGN
serve as alternative receptors for SARS-CoV entry independently
CD147, a surface transmembrane glycoprotein could represent a co-receptor of ACE2 [56–58]. for viral entry as it
CD147,
can enhance a surface
infection by transmembrane
SARS-CoV as well glycoprotein
as HIV-1 could represent
or Measle Virusa [59–61].
co-receptorChenfor et
viral
al. entry
showed as itthat
can enhance
N protein infection bound
of SARS-CoV by SARS-CoV as well asAHIV-1
to Cyclophilin couldor Measlewith
interact VirusCD147
[59–61].[59].
Chen et al.
This showed
protein (also
that N protein of SARS-CoV bound to Cyclophilin A could interact with CD147
known as Basigin) is involved in the expression of matrix metallopeptidases and can interact with [59]. This protein
(also known as Basigin) is involved in the expression of matrix metallopeptidases and can interact
several proteins such as integrin, monocarboxylate transporter proteins, cyclophilins or caveolin-1 [62].
with several proteins such as integrin, monocarboxylate transporter proteins, cyclophilins or
Cyclophilin-CD147 interaction has been involved in the regulation of inflammatory responses and
caveolin-1 [62]. Cyclophilin-CD147 interaction has been involved in the regulation of inflammatory
the pathogenesis of various inflammatory-mediated diseases [63]. However to date, there is no
responses and the pathogenesis of various inflammatory-mediated diseases [63]. However to date,
evidence for CD147 as a direct SARS-Cov-2 binding receptor [64] although the Cyclophilin inhibitor
there is no evidence for CD147 as a direct SARS-Cov-2 binding receptor [64] although the
Alispovir couldinhibitor
Cyclophilin inhibit SARS-CoV2 in vitro
Alispovir could [65].
inhibit SARS-CoV2 in vitro [65].
Vimentin
Vimentin a structural protein may alsoserve
a structural protein may also serve asas aa co-receptor
co-receptor asasititcan
caninteract
interact with
with SARS-CoV
SARS-CoV S S
protein during viral entry [66]. Additionally, the cellular receptor neuropilin-1 (NRP1) expressed on on
protein during viral entry [66]. Additionally, the cellular receptor neuropilin-1 (NRP1) expressed
the olfactory
the olfactory epithelium
epithelium hashas beenshown
been showntotofacilitate
facilitate SARS-CoV-2
SARS-CoV-2 infection
infection[67,68].
[67,68]. The
Theinteraction
interaction
between
between thethe S protein
S protein andand NRP1may
NRP1 mayprovide
provide aa possible
possible pathway
pathwayfor forvirus
virusentry
entry into thethe
into nervous
nervous
system through the olfactory
system through the olfactory system. system.
TheThe virusmain
virus main infected
infected cell-types
cell-typesandand target-organs are described
target-organs in Figurein3, Figure
are described and detailed
3, and
hereafter.
detailed hereafter.

Figure 3. Tropism
Figure and
3. Tropism multiple
and organ
multiple organinjuries
injuriesin
inSARS-CoV-2 infection.SARS-CoV-2
SARS-CoV-2 infection. SARS-CoV-2 infection
infection hashas
beenbeen
associated withwith
associated multiple organ
multiple injuries
organ due to
injuries viral
due to tropism. Among
viral tropism. injured
Among organsorgans
injured (and targeted
(and
cell) we can find: lung (type II pneumocyte), heart (cardiomyocyte), liver (cholangiocyte), spleen and
lymph nodes (macrophage), kidney and brain.
Int. J. Mol. Sci. 2020, 21, 5932 7 of 40

2.3.1. Nasal Cavity

Since SARS-CoV-2 is notably transmitted by droplets, the nasal cavity is its first route of entry.
Among the cells present in the nasal cavity, respiratory epithelial cells (goblet cells and ciliated
cells) were demonstrated to express ACE2 and TMPRSS2 by some [15,17,42] while only TMPRSS2 by
others [69]. Thus, these cells represent an accessible source of viral material for detection of SARS-CoV-2
infection with higher viral load on the nasal swab than on the throat swab [70], meaning that the virus
actively replicates in this place. Therefore, the nasopharyngeal swab is usually employed in routine for
a diagnosis purpose. In addition, these data make these cells a possible reservoir for dissemination
within and between individuals [42].

2.3.2. Olfactory Epithelium

From a clinical point of view, nasal infection of SARS-CoV-2 is associated with olfactory and
gustatory dysfunctions, which should be recognized as important symptoms of infection [71], observed
in more than 50% of the patients [72,73]. Olfactory epithelium was shown to express ACE2 and
TMPRSS2 [74], but also NRP-1 [67,68]. Even if pathophysiology underlying anosmia and gustatory
dysfunction is not clearly elucidated, three mechanisms are evoked [75]. First, local inflammation due
to infection of vascular and support cells of olfactory bulb, provoke downstream effect blocking odor
conduction. Second, damage of support cells, responsible for water and ion balance, could influence
signaling from olfactory sensory neurons. Third, damage to sustentacular and Bowman’s gland cells
may lead to olfactory sensory neurons death and disruption of the entire olfactory epithelium [75].

2.3.3. Lung
Lung is the organ the most concerned by SARS-CoV-2 outcomes. The most common
symptomatology of COVID-19 is related to lung and respiratory tract involvement: dry cough,
sputum production and shortness of breath). Indeed, infection leads to pneumonia (abnormalities on
chest CT) and in severe cases to respiratory failure, which is generally the cause of death [2,9,76–78].
However, the COVID-19 pneumonia is an atypical viral pneumonia, with frequently observed
dissociation between patients’ well-preserved lung mechanics and the severity of hypoxemia [79]. This
discrepancy could be explained notably by lung vascular injury and thrombosis induction leading
to lung hypoperfusion [80]. On a histopathological point of view, pulmonary changes are the most
significant, although nonspecific. In the context of SARS-CoV-2 infection, different histopathological
patterns of acute lung injury have been described so far: diffuse alveolar damage (DAD), which is
closely associated with acute respiratory distress syndrome (ARDS), acute fibrinous and organizing
pneumonia (AFOP) and lymphocytic pneumonia [81–83]. However, the frequency, the context and
the clinical implication of various histopathological patterns is yet-to-be elucidated. DAD pattern,
characterized by numerous hyaline membranes, is often reported, associated with lymphocytes and
few macrophages infiltration, as congested alveolar capillaries [84,85]. Type II pneumocytes are
the principal target of SARS-CoV-2 in the lung, leading to desquamative and dysmorphic pneumocytes
(multinucleated syncytial cells, atypically enlarged, large nuclei, amphophilic granular cytoplasm and
prominent nucleoli) [82]. The main pulmonary pathologic findings are summarized in Table 1.
Int. J. Mol. Sci. 2020, 21, 5932 8 of 40

Table 1. Main pathologic findings in the context of SARS-CoV-2 infection reported in the literature.
DAD: diffuse alveolar damage and AFOP: acute fibrinous and organizing pneumonia.

Organ Main Reported Pathologic Findings

Edema
Mononuclear inflammatory cells (lymphocytes, plasmocytes)
Cellular or proteinaceous exudate
Type II pneumocyte hyperplasia with cytologic atypia
Lungs Vascular congestion
Fibrinoid vascular necrosis
Hyaline membrane formation (hallmark of DAD)
Fibrin deposition with early organization
Extensive intra-alveolar “fibrin balls” and organizing pneumonia (AFOP)
Desquamative pneumocytes
Plasma cell and lymphocyte infiltrate
Gastrointestinal
Edema in the lamina propria
tract
Partial epithelial degeneration, necrosis, shedding of gastric and intestinal mucosa
Mild microvesicular steatosis
Liver Mild lobular and portal activity
Centrilobular sinusoidal dilatation
Cardiomyocyte hypertrophy
Degeneration and necrosis of some cardiomyocytes, mild interstitial hyperemia
Heart
Edema
Infiltration of a small number of lymphocytes, monocytes and neutrophils
Diffuse proximal tubule injuries (loss of brush border, vacuolar degeneration, acute
tubular necrosis)
Kidney
Lymphocyte infiltration
Glomerular injuries

Loss of ACE2 activity after infection has been suggested to be associated with acute lung injury as
it induces an imbalance of the renin–angiotensin system as aforementioned [86].

2.3.4. Gastrointestinal Tract

It is also possible to cite gastrointestinal outcomes including diarrhea, nausea, vomiting and rarely
abdominal pain [87]. SARS-CoV-2 was reported to infect ACE2+ glandular cells in the gastric, duodenal
and rectal epithelia [88]. Histopathology indicated infiltration of plasma cells and lymphocytes and
interstitial edema in the lamina propria, in the context of SARS-CoV-2 infection [85,88]. Moreover, in
some reports, infection in the gastrointestinal tract is associated with partial epithelial degeneration,
necrosis, shedding of gastric and intestinal mucosa [85] and in other, with no damage of the mucous
epithelium [88] (Table 1). Of note, if discharge criteria of patients are mainly based on the resolution of
pulmonary symptoms (with resolved respiratory symptoms, improved acute exudative lesions on CT
images and two consecutively negative RT-PCR from respiratory samples), gastrointestinal persistence
of SARS-CoV-2 may be underrated. Indeed, viral RNA have been detected in stools of some patients
even after resolution of respiratory symptoms and up to 35 days after onset of symptoms, leading
to a protracted form of disease characterized by gastroenteritis symptoms requiring readmission to
hospital [89].

2.3.5. Liver
It is largely reported that aspartate transaminase (AST), alanine transaminase (ALT) and bilirubin
levels are increased during COVID-19, these three markers testifying from liver injury. In addition,
they are often accompanied by an increase of acute phase inflammation markers (procalcitonin and
C-reactive protein) [2,76,78]. This liver injury is usually mild and transient. MERS-CoV was already
associated with liver injury, confined in portal tract and hepatic lobule [90]. These findings were
Int. J. Mol. Sci. 2020, 21, 5932 9 of 40

corroborated for SARS-CoV-2, with mild microvesicular steatosis, mild lobular and portal activity
and centrilobular sinusoidal dilatation [81,82] (Table 1). It has been shown that SARS-CoV-2 is able to
target cholangiocytes. Its cytopathogenic effects might in part explain the liver damages observed
in COVID-19 patients, due to accumulation of bile acid [91]. Apart from viral direct effects, other
mechanisms cannot be excluded, such as drug toxicity.

2.3.6. Cardiovascular System

Patients with pre-existing cardiovascular disease are at higher risk for mortality from
COVID-19 [92]. Moreover, cardiovascular complications are frequent among patients with COVID-19,
regardless of their pre-existing conditions [93,94]. Myocardial inflammation and injury are a common
manifestation of COVID-19 as it has been reported during the MERS-CoV or the SARS-CoV [95,96].
SARS-CoV-2 could infect and induce cytotoxicity in human cardiomyocytes in vitro and recent autopsy
evidences confirmed that direct myocardial tissue infection might occur given that ACE2 is highly
expressed in myocardium [97,98]. On a histopathological plan, some described cardiomyocyte
hypertrophy, degeneration and necrosis of some cardiomyocytes, mild interstitial hyperemia, oedema,
infiltration of a small number of lymphocytes, monocytes and neutrophil [85] while others reported
a few interstitial mononuclear inflammatory infiltrates [82].
The endothelium is also an essential contributor to the pathogenesis of COVID-19 [99].
Histopathology indicated that SARS-CoV-2 might induce endotheliitis in several organs, including
the lung, as a result of both direct endothelial cells infection and overreacting inflammatory
response [100]. Moreover, endotheliopathy characterized by increased thrombotic and microvascular
injuries are also an important feature of COVID-19 complications and are likely to be associated with
critical illness and death [101].

2.3.7. Brain
Regarding the proximity of nasal mucosa, the first entry-site of infection, with the brain (via
the cribriform plate of ethmoid), it is possible to assume an infection of the central nervous system
(CNS). Above all, CNS manifestations were reported during with previous epidemics of SARS-CoV
and MERS-CoV, where CNS manifestations were reported [102,103]. Indeed, SARS-CoV had shown
the ability to infect neurons [104,105]. Consistent with these data, CNS manifestations of COVID-19 are
also depicted (headache, nausea and vomiting) suggesting that CNS is targeted by SARS-CoV-2. Among
severe patients, even acute cerebrovascular diseases and impaired consciousness was observed [106–
108]. However, these manifestations are clearly nonspecific, thus involvement of CNS remains
to be ascertained, notably through cerebrospinal fluid explorations or autopsies [109]. Anyway,
it remains that more specific neurological manifestations were observable in some patients [108].
Thus, Moriguchi et al. reported a first case of meningoencephalitis. This patient initially presented
a generalized seizure and Glasgow score of 6 associated with SARS-CoV-2 RT-PCR positivity in
cerebrospinal fluid but not the nasopharyngeal swab. Additionally, SARS-CoV-2 can lead to spinal
cord injury and myelitis [107,108], which manifested as the Guillain-Barré syndrome notably in two
case reports [110,111]. Finally, a possible involvement of SARS-CoV-2 infection in the development of
long-term neuropathology, such as multiple sclerosis, is conceivable [108]. Indeed, SARS-CoV infection
has already been linked with multiple sclerosis [108].

2.3.8. Kidney
Acute kidney failure was reported during SARS-CoV-2 infection [112,113]. Among the clinical
manifestations of kidney dysfunction, hematuria [114], elevation of serum creatinine [115], blood
urea nitrogen [115] and/or proteinuria [114–116] were reported. SARS-CoV-2 seems to be able
to target the kidney: virus-like particles were visible in the kidney by electron microscopy and
immunohistochemistry showed accumulation of SARS-CoV-2 antigen in kidney tubules. Moreover,
Int. J. Mol. Sci. 2020, 21, 5932 10 of 40

pathologic findings include diffuse proximal tubule injuries (loss of brush border, vacuolar degeneration
and even acute tubular necrosis), lymphocyte infiltration [112] and glomerular injuries [85] (Table 1).

2.3.9. Secondary Lymphoid Organs

On a whole new level, SARS-CoV-2 is targeting the immune system. Indeed, ACE2 was shown to be
expressed by macrophages from lymph nodes and spleen. This has been related to histologic disruption
of these secondary lymphoid organs [85,117]. Furthermore, even if T cells and B cells are not directly
targeted by SARS-CoV-2, it has been shown that SARS-CoV-2 induces lymphocytes apoptosis via
enhancement of Fas-signaling and IL-6 signaling, which may partially explain lymphopenia observed
in infected patients [117], associated eventually to other cytokines-induced phenomena [118,119].

2.3.10. Adipose Tissue

Recent data points to overweight and obesity as major risk factors for developing a severe form of
COVID-19. In a recent French retrospective study nearly half of the patients admitted in an intensive
care unit for COVID-19 had a body mass index—BMI over 30 kg/m2 . In that study severe obesity
was a risk factor to require invasive mechanical ventilation independently of diabetes, hypertension,
sex and age [120]. These data are not surprising as obesity has been previously reported as an
independent factor for severe respiratory form and morbidity in other viral infections such as H1N1
influenza [121,122].
A link between obesity and COVID-19 severity may be related to the adipose tissue. Adipose
tissue can be a direct target of SARS-CoV-2 as the ACE2 receptor is found in adipocytes smooth
muscle cells and endothelial cells within this tissue [123]. However, there is currently no evidence that
the adipose tissue acts as a reservoir for the virus.
Adipose tissue is a highly vascularized and active secreting organ interfering with metabolism but
also immunity. Obesity is considered as a low-grade inflammatory disease due to the altered profiles
of cytokines secreted by the dysfunctional adipose tissue [124]. Obesity is associated with increased
levels of proinflammatory cytokines secreted by adipose tissue as IL-6, IL-1 and TNF-α and a decrease
of anti-inflammatory cytokines such as IL-10. The “pre-activated” adipose tissue may participate in
the explosive proinflammatory state in case of extensive infection. Severe forms of COVID-19 are
associated with a “cytokine storm”, many of the reported cytokines are secreted in other inflammatory
conditions by adipose tissue [125]. Lymphocytes and macrophages dysfunction are common in obesity
with decreased Treg and lymphocytes T suppressor/helper. The same features have been found in
severely ill patients with SARS-CoV-2 [125].

2.3.11. Musculoskeletal Symptoms

Frequent myalgia and fatigue (44–62%) were reported in Chinese and European series [126,127].
The cell receptor gene ACE2 was also shown to be expressed at low levels in human muscular
tissue [128]. Respiratory muscles might also be affected [129]. No joint or bone involvement was
reported so far.
Even with all these data, fundamental research is urgently needed to assess outcomes of
the infection in the different organs, as no consensus exist for organ injury, and knowing that
some histopathological findings cited may be linked either to a direct effect of virus, to an exacerbated
immune response, to pre-existing injuries or even to pharmacological care.
Int. J. Mol. Sci. 2020, 21, 5932 11 of 40

3. Immunopathology of SARS-CoV-2

3.1. Cellular Response

3.1.1. Innate Immune Response

Pattern Recognition Receptor

Innate immune cells need to recognize pathogens (via pathogen associated molecular
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 40
pattern—PAMP) by receptors like pattern recognition receptor—PRR. In coronaviruses, as RNA
viruses, endosomal
viruses, endosomalreceptor Toll Like
receptor Toll Receptor (TLR) 3 (TLR)
Like Receptor and TLR73 andand cytosolic
TLR7 and retinoic acid-inducible
cytosolic retinoic
gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA-5)
acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA-5) havehave been shownbeento
shown to
recognize recognizeand
SARS-CoV SARS-CoV
MERS-CoV. andThis
MERS-CoV. This leads
leads to activation of atosignaling
activation of a signaling
cascade cascade
through NF-κB and
through NF-κB and IRF3 pathways, resulting in a classical immune antiviral response with
IRF3 pathways, resulting in a classical immune antiviral response with induction of type I interferon
induction
(IFN) of type
and other I interferon (IFN)
proinflammatory and other
cytokines as proinflammatory
shown in Figure 4cytokines as shown
[130]. During in Figure
SARS-CoV and4 [130].
MERS
During SARS-CoV
infection, and MERSstrategies
diverse dampening infection,ofdiverse dampening
this cytokine strategies
response haveof this shown
been cytokine response
and haveto
are related
been shown
disease and
severity are related to disease severity [131].
[131].

Figure
Figure 4. 4. Mildversus
Mild versussevere
severeimmune
immuneresponse
response during
during SARS-CoV-2
SARS-CoV-2 infection.
infection.InInregards
regardstotocytokine
cytokine
signature during SARS-CoV-2, mild and moderate cases showed a controlled response withhigher
signature during SARS-CoV-2, mild and moderate cases showed a controlled response with higher
expression
expression of of IL-1β,
IL-1β, IL-1RA,
IL-1RA, IL-2RA,
IL-2RA, IL-6,
IL-6, IL-7,
IL-7, IL-8,
IL-8, IL-9,
IL-9, IL-10,
IL-10, basic
basic FGF,FGF, G-CSF,
G-CSF, GM-CSF,
GM-CSF, HGF,HGF,
IFNγ,
IFNγ,
IP-10, IP-10,MIP-1a,
MCP-1, MCP-1, MIP-1a,
MIP-1b, MIP-1b,
PDGF, TNF-αPDGF,
and VEGF.TNF-α
While,and VEGF. While, aimmunopathological
a cytokine-induced cytokine-induced
immunopathological
mechanism mechanism
has been observed has
with anbeen observed
increase with
of IL-2, an IL-17,
IL-7, increase of IL-2,
IL-10, IL-7, MIP-1a
MCP-1, IL-17, IL-10, MCP-1,in
and TNF-α
MIP-1a
severe andleading
cases, TNF-α into severe cases, effect.
a bystander leading to a bystander effect.

Thisimmune
This immuneevasion
evasionleads
leads to
to aa greater
greater viral
viral load
load by
by delaying
delaying the
thetype
typeI IIFN
IFNresponse
responsethus
thus
following in an exacerbated bystander immunopathogenicity [132,133]. One
following in an exacerbated bystander immunopathogenicity [132,133]. One key component of key component of the
theimmunopathogenesis
immunopathogenesis is the
is theaccumulation
accumulation of macrophages
of macrophagesandand
neutrophils in tissues.
neutrophils in tissues.
SARS-CoV-2 seems to follow the same pathways for recognition and dampeningstrategies,
SARS-CoV-2 seems to follow the same pathways for recognition and dampening strategies,but
but
other mechanisms may be discovered [130,134]. The major point is the suppression of the type I IFN
other mechanisms may be discovered [130,134]. The major point is the suppression of the type I IFN
response [130,135,136]. Although monocytes/macrophages and dendritic cells should theoretically
response [130,135,136]. Although monocytes/macrophages and dendritic cells should theoretically be
be key producers of type I IFN, as primary responding tissue innate immune cells, studies to date
key producers of type I IFN, as primary responding tissue innate immune cells, studies to date showed
showed minimal cytokine expression in the first stages of the disease.
minimal cytokine expression in the first stages of the disease.
Dendritic Cells (DCs)
As the strongest antigen-presenting cells (APC), dendritic cells (DCs) play a leading role in
stimulating and linking innate and adaptive immunity. They represent one of the first-line cells
during the onset of infectious disease. Thus, they are a direct target of viral evading strategies to
reduce the immune response. Studies on DCs susceptibility for other coronaviruses showed
susceptibility to SARS-CoV but at a low level and without a strong triggering of type I IFN secretion.
Int. J. Mol. Sci. 2020, 21, 5932 12 of 40

Dendritic Cells (DCs)

As the strongest antigen-presenting cells (APC), dendritic cells (DCs) play a leading role in
stimulating and linking innate and adaptive immunity. They represent one of the first-line cells during
the onset of infectious disease. Thus, they are a direct target of viral evading strategies to reduce
the immune response. Studies on DCs susceptibility for other coronaviruses showed susceptibility to
SARS-CoV but at a low level and without a strong triggering of type I IFN secretion. On the other hand,
MERS-CoV was able to activate DCs and to induce a greater cytokine reaction than SARS-CoV [137].
With the presence of ACE2 on the surface membrane of DCs, a question remains on their infectability
by SARS-CoV-2 [138]. As major producers of type I IFN, they also play a role in cytokine release
and attraction of immune cells. Comparably to SARS-CoV, the induction of such cytokine during
SARS-CoV-2 infection is to be further explored [134]. In COVID-19 patients, DCs percentage in blood
did not change with disease severity but they showed a reduction of CD86 expression in severe
compared to mild disease [139]. Variation of DC subsets has been observed. CD1c+ DCs were
decreased with disease severity and recruited in the lung during ARDS [140].

Macrophages
Monocytes and macrophages are important innate immunity actors of airway interface. In
pulmonary infection, alveolar macrophages initiate the production of type I IFN [141].
Alveolar macrophages express ACE2 but at a low level [138]. They are present since the initiation of
the disease and represent one of the initial cells involved in the innate immune response. The mechanism
of entry of the virus in macrophages is still under study: through phagocytosis, ACE2-dependent [117]
or -independent pathways. Studies of the SARS-CoV and MERS-CoV cellular response showed
a delayed cytokine response in the initial stages of the disease. Later, they are responsible for
releasing high levels of proinflammatory cytokines but low amounts of IFNs. However, the IFN
response in the initial stages of viral infection is key to mounting antiviral immunity. The secondary
rapid cytokine release by recruiting proinflammatory cells is hypothesized to be responsible for
host tissue injury [142]. During COVID-19, a decrease of classical monocytes (CD14+ and CD16−)
was observed with an increase in intermediate (CD14+ and CD16−) and non-classical (CD14− and
CD16+) monocytes [141]. A mouse model of SARS-CoV infection showed that a delayed type I
IFN release increased disease mortality through accumulation of inflammatory macrophages [143].
Depletion of those macrophages, but not of neutrophils, led to a protective effect on disease mortality
without an increase in viral load [143]. In bronchoalveolar lavage of COVID-19 infected patients,
monocytes-derived macrophages (overwhelming tissue resident macrophages) and neutrophils were
increased in severe patients. Additionally, macrophages had an M1-like gene expression pattern in
severe patients whereas moderate patients had a more M2-like macrophage profile [144]. Type I IFN
can induce macrophages polarization depending on their microenvironment [141]. During severe
COVID-19, macrophages have a M1 proinflammatory cytokine profile and are likely to be a key factor
of cytokine related severity clinical features [145,146].

Polymorphonuclear Neutrophils (PMN)

Neutrophils are predominant actors for disease control in innate immunity specifically during
respiratory diseases and ARDS. Neutrophils have a central role in driving an inflammatory pathogenic
state, with the production of reactive oxygen species (ROS), cytokines, proteases and neutrophil
extracellular traps (NETs), which exude histones and DNA [147–149]. NETs are key features to
contain infection but some pathogenic effects have also been described [150,151]. As ARDS and
immunothrombosis are predominant features in critically ill patients in COVID-19, the implication of
neutrophil in this setting has to be explored. In previous coronaviruses infections, neutrophil appears
as a major actor of aggravation rather than disease control. An animal model of depleting neutrophil
during coronavirus infection shows an essential role of PMN for mounting an effective viral response
Int. J. Mol. Sci. 2020, 21, 5932 13 of 40

but induced lung injury [152]. An increase in neutrophil count has been associated with disease
severity. Single-cell RNA sequencing based peripheral blood mononuclear cells profiling showed an
increased neutrophil subpopulation expressing a progenitor profile in severe cases consistent with
the reported neutrophil expansion [153]. Furthermore, the reduction of the ratio of neutrophil to
lymphocyte is associated with a worse outcome [154]. A recent study of NETs during COVID-19
showed an increased sera level of cell-free DNA, myeloperoxydase-DNA and citrullinated histone
H3. These were increased in critical patients needing mechanical ventilation compared to those not
mechanically ventilated [155,156]. This role of NETs in ARDS may represent a new therapeutic target
in those settings [148,155]. At a histological level, neutrophil infiltration has been confirmed in autopsy
samples [155].

Complement
The complement system plays an essential role during response to bacterial, viral and fungal
infection. It acts rapidly to activate the host immune system and attract cells towards the infection site.
Its activation is tightly controlled to avoid detrimental injury. C3a and C5a have proinflammatory
properties by recruiting inflammatory cells and activating PMN. They participate in acute pulmonary
injury during coronavirus infection [157]. The use of an anti-C5a antibody in a mouse model of
MERS-CoV infection has shown an improvement of respiratory symptoms without an increase in viral
load [157].
A recent histological study of pulmonary and cutaneous biopsies or autopsy samples from five
critically ill patients infected by SARS-CoV-2 showed complement mediated microthrombotic disease
with deposits of C5b-9, C4d and Mannan-binding lectin serine protease 2 (MASP2). Thus, some atypical
ARDS features of COVID-19-related severe respiratory distress might be caused by microvascular
injury after activation of complement pathways [158]. The complement system seems a good candidate
for a therapeutic target to limit disease severity by limiting inflammatory cell recruitment or direct
complement activation related microthrombopathy [134,158].

Natural Killer (NK) Cells

With CD8+ T cells, NK cells are predominant actors to impeding viral replication by killing
infected host cells. In previous studies on SARS-CoV, a dampening of cell-mediated cytotoxicity was
observed with a reduction of NK and CD158+NK cells with disease severity [159]. Animal models of
SARS-CoV infection with specific NK cell depletion found no aggravation of the disease suggesting
that NK cells are not essential for the SARS-CoV immune response [102,159]. Clinical studies during
COVID-19 showed a decrease of NK cell count in severe but not mild disease, probably linked to
viral propagation [146,153,154]. The dynamic profile of the NK cell count did not show differences
between dead or survivor groups of the most severe patients [160]. The time to NK cell normal cell
count recovery paralleled clinical amelioration. However, SARS-CoV severe disease was marked by
a functionally exhausted profile with increased membrane expression of NKG2a and a reduction of
CD107a+, IFNγ+, IL-2+ and TNF-α+ NK cells [161]. Wilk et al. demonstrated that NK CD56+ cells
were reduced in all COVID-19 patients and NK CD56- cells were reduced in ARDS patients [153].

3.1.2. Adaptive Immune Response

T Cells
Secondarily to the innate immune cells initial reaction, the adaptive immunity plays an important
role in recognition and killing of infected cells with cytotoxic CD8+ lymphocyte and in mounting
a humoral antigen specific response with CD4+ lymphocyte. Dysregulation of lymphocyte subsets
can occur during viral infection. During COVID-19, lymphopenia is a common feature [162]. A
decrease of total lymphocytes but also of CD4+ T cells, CD8+ T cells and B cells decrease was reported.
The lymphocyte levels were lower in severe compared to mild cases. This is comparable to data
Int. J. Mol. Sci. 2020, 21, 5932 14 of 40

during SARS or MERS infection. In addition, CD8+ T-cell decrease and CD4/CD8 ratio increase were
correlated with disease severity [162]. The normalization of the different subset cell counts appeared
during the clinical convalescence.
In the context of previous coronaviruses epidemics, a strong T-cell response was associated with
higher titers of neutralizing antibodies. Specific T-cytotoxic cells were still found 10 years after infection
with SARS or MERS infections [163–166].
During SARS infection, the viral structural proteins E [167], S [168], M [169] and N [167,170]
were shown to induce T cell antigen-specific responses [165,166]. The acute phase is followed by
an important contraction of the antigen-specific T cell pool. T CD4 antigen specific cells presented
a central memory phenotype (CD45RA− CCR7+ CD62L−) with production of IFNγ, TNF-α or IL-2
whereas CD8+ T cells presented an effector memory one (CD45RA+ CCR7− CD62L−) with production
of IFNγ, TNF-α, perforine and granzyme [59,165,166]. Emerging data point that memory CD4+ T cell
specific for commonly circulating human coronaviruses (HCoV-OC43, HCoV-229E, HCoV-NL63 and
HCoV-HKU1) can cross-react with SARS-CoV-2 epitopes [171]. Thus, Mateus et al. mapped 142 T cell
epitopes across the SARS-CoV-2 genome and showed a population of responsive memory CD4+ T
cells, even in unexposed individuals [171]. Even if it is still very speculative, this pre-existing repertoire
of memory CD4+ T cell against other coronaviruses may cross-react with SARS-CoV-2 and contribute
to variations observed in COVID-19 patient disease outcomes [171–173]. This hypothesis is supported
by similar data, indeed, reactivity in blood samples of unexposed individuals has been reported by
several teams [174–178].

B Cells
The importance of mounting the humoral response was shown by the presence of a non-switched
B lymphocyte profile in deceased patients from SARS or MERS viruses. The role of neutralizing
antibodies is essential in a later stage of the disease and to prevent reinfection.
Evaluation of the antibodies response after SARS-CoV infection showed apparition of IgG as soon
as 4 days after infection. Most patients had a seroconversion at 15 days [163,179]. Antibodies directed
against the coronavirus S protein are protective neutralizing antibodies but binding non-neutralizing
antibodies against other viral proteins are also described [180]. During the follow-up period, IgM
peaked at 1 month and IgG at 2–4 months [181]. A decrease of antibody titer was observed at 3
years [181] and undetectable in 21 of 23 patients at 6 years [182]. There was no association between
the drop of neutralizing antibody titer and disease severity, comorbidity or treatment use [183].
In SARS-CoV-2, the same chronological appearance of IgM and IgG has been observed [184]. In
severe cases, a strong acute humoral response level has been reported [185] with increased proportion
of plasmablasts in COVID-19 patients [153]. Nevertheless, a rapid decrease of the SARS-CoV-2 specific
antibodies titers has been demonstrated in severe cases [186–188]. Variation of antibody response
kinetic with disease severity [188,189], anti-N and anti-S antibodies antigen specificity [187,189],
cytokine environment variation or major lymphopenia with disease severity [190] has been described.
Their impacts on humoral protection maintenance have to be further studied. Long-lived plasma
cells and memory-B cells responses and maintenance are important actors of long-term humoral
protection [191,192]. The mechanisms underlaying the lack of long-term humoral immunity in some
infectious disease are poorly understood [193]. Pathogen’s antigen evasion mechanism [194], lower
B-memory cell response [195,196] or long-lived cell homeostasis anomaly [197,198] may be leads
to explore.
In some animal and human coronaviruses, the mechanism of antibody-dependent enhancement
(ADE) has been suspected comparably to the dengue virus. Notably for SARS-CoV, the in vitro
experiment showed enhanced infection with specific anti-spike antibodies highly diluted [199,200].
Based on the similarity between SARS-CoV and SARS-CoV-2, some question the possibility of
occurrence of ADE during COVID-19 [201] but to date no strong evidence has been found [202].
Int. J. Mol. Sci. 2020, 21, 5932 15 of 40

Post-infectious autoimmune disorders have been reported [203,204] such as antiphospholipid

syndrome [205], immune thrombocytopenic purpura [206,207], autoimmune hemolytic anemia [208,
209], Guillain-Barré syndrome [110,210,211] or Kawasaki-like syndrome in children [212].

3.2. Molecular Response

To mount an effective antiviral response, innate and adaptive immunity are essential to control
viral infection and clean infected cells with limited inflammation. For their actions immune cells
release variable panels and levels of cytokines. This release if uncontrolled can have detrimental
consequences for the patient. A delayed response may induce viral tissue injury, which can in turn
lead to an exaggerated proinflammatory cytokine response. In the setting of sepsis and multi-organ
dysfunction syndrome (MODS), two phases are described. An early phase with systemic cytokine
release, neutrophil-generated ROS and cytotoxic lymphocyte inducing inflammatory systemic response
with endothelial damage. A later phase consists of an amplification of inflammation by interstitial
compartment cells in organs further exacerbating parenchymal injury [213].
The pulmonary failure during MODS appears as acute lung injury or ARDS (Figure 4). In
pathogenesis, cytokines act to initiate and amplify the inflammatory response. Some major cytokines
found are IL-1, IL-6, IL-8, IL-10 and TNF-α. The cytokine attraction and activation of neutrophils acts
as the main pathogenic system inducing epithelial lung injury. This was confirmed with histological
studies [213].
ARDS and immunothrombosis are predominant features in severe COVID-19 [142,214]. It has
been long described the importance of neutrophil activation with NETosis, cytokine, ROS and protease
release in pathophysiology of viral ARDS and immunothrombosis in sepsis [147–149,215,216].
In SARS-CoV infection, the viral 3a protein has been shown to induce NLRP3 mediated
inflammasome activation [217]. A delayed innate immune response with low type I IFN release was
associated with a later exacerbated proinflammatory (such as IL-1 and IL-6) cytokine response in severe
cases. The timely cytokine response is necessary for a rapid recruitment and clearance of neutrophils
and transition to adaptive immunity with minimal immune tissue damage [218].
In regards to cytokine signature during SARS-CoV-2, studies showed an adapted, controlled
response in mild and moderate cases with higher expression of IL-1β, IL-1RA, IL-2RA, IL-6, IL-7, IL-8,
IL-9, IL-10, basic FGF, G-CSF, GM-CSF, HGF (hepatocyte growth factor), IFNγ, IP-10, MCP-1, MIP-1a,
MIP-1b, PDGF, TNF-α and VEGF compared to a healthy control as shown in Figure 4 [2,126,219]. These
results are summarized and compared to other infectious settings in Table 2 [126,220–224].
In severe cases, clinical features show severe pneumonia, ARDS or MODS [2]. A cytokine-induced
immunopathological mechanism has been observed with an increase of IL-2, IL-7, IL-17, IL-10, MCP-1,
MIP-1a and TNF-α compared to moderate cases [126] (Figure 4). Other studies looking for cytokine
biomarkers of worse prognosis between severe and moderate cases show that most cytokines were
comparable between groups [2,219]. However, IP-10, MCP-3 and IL-1RA were significantly higher
with a correlation to gravity evaluated by PaO2/FiO2 and Murray scores [219]. TNF-α, IL-1, IL-8 and
IL-6 also increased in severe vs. non severe cases [220]. TNF-α has been shown to enhance ACE2
shedding, which participate to lung injury during SARS-CoV [145]. There was no increased risk of
severe COVID-19 for patients treated by TNF-α blockers [225]. A case of severe COVID-19 disease
in a recently diagnosed Crohn’s disease was ameliorated under TNF-α blockade [226]. Similarly to
SARS-CoV, pyroptosis may be triggered during COVID-19 leading to major IL-1β production and
uncontrolled cytokine release [145,217]. An increased IL-6/IFNγ ratio, found in severe cases, could be
a sign of inappropriate immunopathologic cytokine response [218].
Int. J. Mol. Sci. 2020, 21, 5932 16 of 40

Table 2. Cytokine profile during diverse infections.

COVID-19 Severe
Cytokine Dengue Fever Leptospirosis
[220] * Chikungunya Viral Sepsis [222] Bacterial Sepsis [222]
(pg/mL) [221] [223]
[126] # [224]
IFNγ 9# 772.4 ± 1762.7 12.6 ± 10.8 9.54 ± 19.16 7.2 16.8 ± 39.61
IL-1 5 (no detectable) * 3.3 ± 2.2 209.6 ± 936.8 9.6
IL-2 8# 2.5 ± 0.8 1.46 ± 1.04 4 3.2 ± 6.67
IL-6 25.2 (9.6–54.5) * 24 ± 47 671.9 ± 1261.3 63.23 ± 265 74.7 2533 ± 6559
IL-8 18.4 (11.3–28.4) * 21.1 ± 10.1 734.1 ± 1721.9 30.31 ± 38.01 251.1 1249 ± 6944
IL-10 6.6 (5–11.3) * 32.5 ± 54 90.9 ± 417 14.27 ± 16.75 21 205.11 ± 741.85
TNF-α 8.7 (7.1–11.6) * 15.1 ± 107.1 4
IP-10 >1000 # 56.6 ± 278.5 291.8 ± 460 1944 ± 1295 666.72 ± 766
MCP-1 50 * 389.6 ± 929.3 545.4 ± 418.9 494.31 ± 679 4533 ± 1458
MIG 791.7 ± 434.9 660.71 ± 661 1377 ± 1906
IFNγ: Interferon γ; IL-1: Interleukin 1; IL-2: Interleukin 2; IL-6: Interleukin 6; IL-8: Interleukin 8; IL-10: Interleukin 10; TNF-α: Tumor Necrosis Factor α; IP-10: Interferon gamma-induced
protein 10; MCP-1: Monocyte Chemoattractant Protein 1; MIG: Monokine Induced by Gamma interferon. * Data coming from [220]. # Data coming from [126].
Int. J. Mol. Sci. 2020, 21, 5932 17 of 40

Those deregulatory cytokine features may represent potential therapeutic targets for severe
COVID-19. The family of dysregulated cytokine syndromes is vast and often associated with critical
life-threatening features. It is generally simplified as “cytokine storm” but comprise of a wide
range of primary or secondary etiologies [227]. Familial hemophagocytic lymphohistiocytosis (HLH),
macrophage activation syndrome (MAS) a secondary HLH complicating an underlying autoimmune
disease, MAS-like sepsis, which appears in severe sepsis with MODS, are classical representatives of
the “cytokine storm syndrome” [228].
In comparison to primary and secondary HLH, the viral immunosuppression during severe
COVID-19 is supposed to arise from blood lymphopenia [126], NK and CD8 exhausted profiles [161]
and interferon suppression [214,229]. Following the primary delayed response, a secondary excessive
cytokine non-type I IFN but proinflammatory profile (IL-6, IL-1, IP-10, IL-18, GM-CSF and IFNγ)
appears [146,214,219]. Furthermore, an increased and persistent viral release draws a cytokine and
T-cell response to clear infection but induces, at the same time, pulmonary damage and ARDS. This
offers explanation to clinical features typically found in severe COVID-19 pneumonia [214].
Some particularities of severe COVID-19 differ from HLH or MAS. The immune deregulation
seems more confined to the lung with features of ARDS, which is linked to hypercytokine release by
macrophages, attraction and activation of neutrophils [214,215,229].
The typical ARDS features found in severe COVID-19 has a cytokine profile comparable to
H1N1(2009) infection where elevation of IL-6, IP-10 was observed in severe but not mild disease [230].
Compared to other pathological conditions (H1N1 influenzae and bacterial infection), patients with
COVID-19 experiencing ARDS showed lower global severity, as assessed with lower SOFA score. This
suggests an earlier respiratory failure in COVID-19 [146,231].
In many studies, sepsis is used to understand severe COVID-19 physiopathology. A study trying
to classify COVID-19 patients into three immune states found during sepsis (MAS-like, dysregulation
with immunoparalysis or functional) showed that all severe cases had an immune dysfunction
with the majority of patients harboring dysregulated immunity while the rest presented MAS-like
profile [146]. To confirm this classification, a specific severity score (H-score) was increased in MAS-like
severe COVID-19 patients but not in the dysregulated group with immunoparalysis. In the latter
group, serologic measures showed no measurable levels of IFNγ and an increase in IL-6 and CRP
levels. IL-6 being one of the drivers of immunopathogenesis, a specific therapy was tested but showed
only partial efficacy. Further studies need to be produced.
Another predominant cytokine driver of immunopathogenesis described during viral ARDS
is IP-10 [229]. Chemoattractant cytokines for neutrophils overproduced by macrophages in lung
injury are also found during severe COVID-19. They represent potential therapeutic targets for severe
cases [219].

3.3. Immune Evasion

Coronaviruses are well armed to evade the immune system and particularly the initial innate
immune response. The mechanism of molecular immune evasion is still to be confirmed for SARS-CoV-2
but the experience of previous SARS and MERS coronaviruses points some potential ways of immune
evasion [130,232]. A common mechanism to avoid recognition of the virus is the formation of
a double vesicle shielding from PRR sensing [232,233]. Among the structural proteins that serve
in the architecture of the virion, SARS N protein is suspected to interfere with RNA recognition by
the host immune sensors [232,234–236]. SARS and MERS protein M interact with TRAF3 to disrupt
the TRAF3-TBK1 complex [232,237].
MERS non-structural protein 4a (NSP4a), 4b (NSP4b) and papain-like protease (PLpro) are
described to have an immunity-evading function [232,234,237]. NSP4a interacts with PACT to inhibit
dsRNA recognition and inhibit protein kinase R (PKR) [232,237–239]. NSP4b antagonizes OAS-RNAse
L pathway thus limiting RNAse L activation and binds to TBK1 [232,237,240]. NSP15 also limits RNA
Int. J. Mol. Sci. 2020, 21, 5932 18 of 40

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 40

sensor activation but through unknown pathway. PLpro protease uses deubiquitination to inhibit
deubiquitination
ISG15 to inhibit ISG15
action [237]. Additionally, action
NSP 1 in[237].
SARSAdditionally, NSP 1 in host
and MERS degrade SARSmRNA
and MERS degrade host
[237,241,242].
mRNA [237,241,242].
For SARS, NSP 7, 16 and 15 and accessory proteins ORF3b, ORF6 and ORF9b have also inhibitory
ForNSP16
activities. SARS, prevents
NSP 7, 16RNA and sensing
15 and accessory
by MDA5proteins ORF3b, ofORF6
by formation and ORF9b[243].
cap structures have also
ORF9b
inhibitory activities. NSP16 prevents RNA sensing by MDA5 by formation of cap structures [243].
acts by inducing a proteosomal degradation of MAVS/TRAF3/TRAF6 [244]. The others dampen
ORF9b acts by inducing a proteosomal degradation of MAVS/TRAF3/TRAF6 [244]. The others
IFN production through unclear mechanisms but ORF3b and ORF6 alter IRF3 activation and thus
dampen IFN production through unclear mechanisms but ORF3b and ORF6 alter IRF3 activation
antagonize IFN production and signaling through IFNβ notably [232,234]. Furthermore, SARS-CoV
and thus antagonize IFN production and signaling through IFNβ notably [232,234]. Furthermore,
PLpro inhibits PLpro
SARS-CoV IRF3 activation and activation
inhibits IRF3 deubiquitininate RIG-I, TBK1 and
and deubiquitininate IRF3
RIG-I, [232,245].
TBK1 Those
and IRF3 potential
[232,245].
mechanisms for SARS-CoV 2 immune evasion are summarized in Figure 5.
Those potential mechanisms for SARS-CoV 2 immune evasion are summarized in Figure 5.

Figure
Figure 5. Viral
5. Viral sensing,
sensing, innateantiviral
innate antiviralresponse
response and
and immune
immune evasion.
evasion.Potential mechanisms
Potential mechanisms of of
SARS-CoV-2 immune evasion based on previous studies on MERS-CoV (brown)
SARS-CoV-2 immune evasion based on previous studies on MERS-CoV (brown) and SARS-CoV (blue). and SARS-CoV
(blue).
Some Some mechanisms
mechanisms are inhibiting
are inhibiting viral sensing,
viral sensing, whereaswhereas others
others are are directed
directed againstagainst the innate
the innate antiviral
antiviral response. Solid arrows correspond to a direct promoting activity, dotted arrows
response. Solid arrows correspond to a direct promoting activity, dotted arrows correspond to an correspond
to an indirect promoting activity and T-bars correspond to a direct inhibitory activity.
indirect promoting activity and T-bars correspond to a direct inhibitory activity.

Other
Other potential
potential mechanismsare
mechanisms are aa reduction
reduction of
of presentation
presentationtotoananadaptive
adaptiveimmune cellcell
immune by by
downregulation of major histocompatibility complex (MHC) expression on antigen-presenting cells,
downregulation of major histocompatibility complex (MHC) expression on antigen-presenting cells,
Int. J. Mol. Sci. 2020, 21, 5932 19 of 40

viral mutation, immune cell exhaustion and immune deviation to the Th2 profile or localization in
the immune privilege site [130,246].

4. Regards to Discrepancies in Diverse Immune Conditions

4.1. Elderly
During COVID-19 epidemics, studies identified age as a major risk factor of disease severity
and mortality. A higher proportion of severe, critically ill patients or deaths appeared within this
population [247]. The progression to severity was also shorter. Some symptoms (shortness of breath
and lymphopenia) and comorbidities (cardiovascular disease, chronic obstructive pulmonary disease
and acute respiratory distress syndrome) were predictive of worst outcomes [247].
An explanation to this excess mortality is the mounting number of comorbidities with age. A
reduction of T-cell diversity appears with aging. Interestingly, naive T-cell diversity is strongly reduced
after 65 years old [248]. However, modification of senescent T-cell profile appears to compensate this
lack of diversity and replication with aging [248]. Lega S. et al. hypothesized that disease severity with
aging may be due to immune senescence with an insufficient pool of specific T-cell to overcome the viral
replication [249]. In regard to host immunity during COVID-19, the part of the immunosenescence
lacking an antiviral response is to be further explored.

4.2. Children
Since the beginning of the epidemic, children seem to be spared from infection by SARS-CoV-2 [250].
In February, a Chinese cohort found only 2.4% of patients under 19 years old. Among these,
they had 2.5% of the severe forms and 0.2% of critically ill patients, compared to 13.8% and 6.1%
respectively in the whole cohort [251]. They appear to more often suffer from gastrointestinal
symptoms [250]. The lesser potency in children was also observed during SARS-Cov and MERS
disease [250]. The infection mostly occurs through household contact. The immune system shows
some discrepancies between early and adult life in innate and adaptive immune cells. This results
in a difference in response to the pathogen including viruses with some being more potent during
infancy [252]. For SARS-CoV-2, the immune response seems favorable in childhood compared
to adulthood.
Nevertheless, the appearance of a pediatric multisystem inflammatory syndrome has been
reported with clinical features of myocarditis and Kawasaki-like disease after severe SARS-CoV-2
infection [212,253,254].

4.3. Pregnancy
In regard to infectious disease and especially respiratory illnesses, pregnant women represent
a high-risk population. The same has been reported with SARS-CoV and MERS-CoV [255]. For
SARS-CoV-2 infection, an increased risk for preterm delivery, preeclampsia, premature rupture
membrane or stillbirth seem to appear [256] but the mother prognosis seems better than during
SARS-CoV or MERS-CoV diseases [255]. Mortality and morbidity during pregnancy are still unclear.
Some specific elements may explain such over-risk. Firstly, some common gestational symptoms
may mask initial, non-severe COVID-19 symptoms like rhinitis or dyspnea. Thus, leading to community
transmission and late diagnosis. Furthermore, reduction of total lung capacity with advancement of
pregnancy can predispose for respiratory failure [255].
Secondly, focusing on the immune system, there are changes in innate and adaptive immune
responses in the face of an infectious challenge like strong NK cells or monocytes responsiveness [256]
or a shift of the Th1 cytokine profile to a Th2 profile [255]. The pregnancy is associated with global
viral susceptibility. It is to note that, differently to SARS-CoV, the cytokine profile during SARS-CoV-2
infection shows Th1 but also Th2 (IL-4 and IL-10) cytokine increase. The Th2 shift profile in pregnant
women may thus serve to reduce severity [255]. Additionally, the cytokine release occurring during
Int. J. Mol. Sci. 2020, 21, 5932 20 of 40

severe COVID-19, in addition to the gestation-linked proinflammatory state during the first and third
semester, can alter fetal development and explain poor pregnancy outcomes [256].
In regard to their fetuses, no warning signal of vertical transmission has been observed [255].

4.4. Men/Women Susceptibility

A recent review of clinical and epidemiological findings during COVID-19 shows a 3-fold mortality
increase risk in males compared to females [257]. This observation was also reported during SARS-CoV
and MERS-CoV epidemics [258]. An animal model, with mice infected by SARS-CoV, reproduced this
sex bias, which increased with age [258]. Sex hormones were associated to female protection.
In living organism, gender is described to influence an immunological response and affect innate
and adaptive immunity alike [259]. It impacts apparition of autoimmune disease but also responses to
infectious diseases. Furthermore, this influence changes over the course of life [259].
A recent review of current knowledge on sex difference of innate immune cells during respiratory
virus infection showed a variety of receptors levels depending on cell subset and tissue localization [260].
Furthermore, they may act directly to promote or reduce cell function but also can act indirectly by
modulating non-immune cell responses [260].

4.5. Genetic Susceptibility

Clinical aspects of COVID-19 disease are highly variable ranging from asymptomatic to
life-threatening multi-organ failure disease. Differences in host genetic susceptibility may explain
part of those differences in response to viral infection [261]. Host genetic polymorphisms have been
associated with clinical severity in viruses [262,263]. During the SARS-CoV outbreak, HLA [264] and
ABO [265] groups have been associated with disease severity. Using a genome-wide association study
during the SARS-CoV-2 pandemic, HLA risk polymorphisms were described [261,266]. A susceptibility
locus at chromosome 3p21.31 (spanning SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1 genes)
has notably been observed [266]. The ABO group seems to be implicated with a lesser risk of infection
in the O group and higher risk in the A group [266–268]. Further studies may bring new genetic
susceptibility association.

5. Immunotherapy
No antiviral therapy is available for treatment of COVID-19. Vaccines are under intensive
development, and numerous clinical trials have been initiated, this could be the subject of a full-fledged
review. For this reason, we will focus on SARS-CoV-2 management due to immunotherapy.

5.1. Antibody-Based Therapy

The first approach would be to use an old trick (i.e., serum therapy or serotherapy) invented
by Emil Behring, more than 100 years ago, to prevent diphtheria. The purpose is to use serum from
individuals who recovered from COVID-19 to treat suffering patients, the recovered serum containing
neutralizing antibodies directed against SARS-CoV-2 creating passive immunity against the virus.
Serotherapy, intravenous Ig (IVIg) or therapeutic plasma exchange (TPE) has been mentioned by
several teams [269–271] and could be envisaged in prophylaxis or treatment, even if prophylactic
injection or injection early after symptoms onset give better results than later injection [272,273]. Unlike
vaccine therapy, serotherapy gives an immediate but short-lasting immunity (from weeks to months
according to amount and composition) to susceptible persons. Specific antibodies are essential in viral
clearance. Thus, convalescent plasma use may bring direct antiviral effect. Additionally, serotherapy
has immunomodulatory effects that can be helpful in severe COVID-19 [274,275]. This solution has
been used during the SARS-CoV outbreak and showed good results [276,277]. This therapy needs to
be evaluated by clinical trials, taking in account all the challenges this practice could be facing [278].
Notably the potential risks could be: infection with another pathogen due to injection, reaction with
serum components, ADE as a consequence of subneutralizing antibodies could thus facilitating entry
Int. J. Mol. Sci. 2020, 21, 5932 21 of 40

of SARS-CoV-2 in cells [269]. Preliminary studies in severe cases show a reduction of mortality after
convalescent plasma use [279] and need to be further explored.
Polyvalent IVIg, another blood derived product, does not contain neutralizing antibodies against
SARS-CoV-2 and preparation/availability of specific IVIg from convalescent donor may take time [274,
280]. Efficacy of IVIg during SARS-CoV has been variable [280]. Recent clinical trials seem to show an
efficacy of high-dose IVIg in severe patients during COVID-19 [281,282]. Additionally, IVIg has been
useful to treat autoimmune complication due to immunomodulatory effects [283,284].
As shown in sepsis, plasmapheresis was evoked as adjunctive treatment in severe COVID-19 [271,
285] to control the dysregulated immune response but its efficacy is still under question [286,287].
A recent case series showed improved outcomes after plasmapheresis [288]. Additionally, it has
been proposed to use convalescent plasma for a better efficacy of plasmapheresis [289]. Therapeutic
evaluation through randomized trials is still needed.
Another aspect of antibody-based therapy could be the use of recombinant antibody targeting
spike protein of SARS-CoV-2. Several antibodies have been described to target SARS-CoV-2 S. CR3022,
which was initially isolated from a SARS-CoV convalescent patient showed ability to cross-react with
a highly conserved cryptic epitope of S [20]. 47D11 showed the same ability [290]. Additionally,
immunoglobulin fragment F(ab’)2 against RBD produced in horses exhibited a potent capacity to
neutralize SARS-CoV-2 in mice [291]. Similarly, 2B04 a murine monoclonal antibody that binds RBD
showed a potent neutralization of SARS-CoV-2 on the mouse model of infection, reducing weight
loss and viral burden [292]. Of further note, a recombinant extracellular domain of ACE2 fused
to the Fc region of human IgG1 (called ACE2-Ig) showed high affinity for SARS-CoV-2 RBD, and
could be considered for treatment [293]. Interestingly, Gammunex® and Flebogamma® , which are
currently available polyvalent IVIg, showed cross-reactivity with different coronaviruses, including
SARS-CoV-2 [294]. Recently, the risk of escape mutant generation was raised under individual
Spike-specific antibody selective pressure in vitro. This was prevented due to an antibody cocktail,
against two non-overlapping regions of SARS-CoV-2 spike protein, which represents a therapeutic
avenue limiting emergence of escape mutants and subsequent loss of drug efficacy [295].

5.2. Immunomodulators
A viable therapeutic treatment against COVID-19 is urgently needed. Drug repositioning is a fast
way to produce therapeutic treatment. Numerous trials assessing antiviral or immunomodulatory
efficacy of different treatments are ongoing [296].
An initial focus has been made on hydroxychloroquine, a derivative of chloroquine.
Hydroxychloroquine is a known antimalarial and immunomodulatory molecule actually used for
treatment during autoimmune diseases such as systemic lupus erythematosus (SLE). It acts by increasing
pH in endosomes thus interfering with the antigen processing of antigen presenting cells (APC) [297].
An antiviral effect of chloroquine on both entry and post-entry stages of SARS-CoV-2 infection has been
described in vitro, which could add to its immunomodulatory effect in vivo [298]. Potential mechanisms
are not fully understood. Presumably, the increase of pH in endosomes and drug accumulation in
lysosomes prevent viral entry and transport [299]. Furthermore, an antiviral action during SARS-CoV
infection was shown by interfering with the terminal glycosylation of ACE-2 [300] and glycosylation
of viral particles [301]. A review of clinical trials and cohort study of hydroxychloroquine showed
conflicting results on efficacy and viral clearance [302–304]. Adverse events of hydroxychloroquine are
long known. The most serious adverse events are ocular (retinopathy) following chronic exposition,
cardiovascular (restrictive or dilated cardiomyopathy and conduction abnormalities), cutaneous
(alopecia, pruritus, photosensitivity and skin eruption), neurologic (headache, dizziness, vertigo,
convulsion and polyneuropathy), hepatic (abnormal liver function and fulminant hepatic failure)
and gastrointestinal (diarrhea, anorexia, nausea and vomiting) [297,305]. In regard to the use of
hydroxychloroquine during COVID-19, studies seem to show a relatively safe profile [299,306].
Int. J. Mol. Sci. 2020, 21, 5932 22 of 40

Its efficacy being still not demonstrated, it was removed from treatment guidelines until further
studies [307,308].
Systemic corticosteroids have a broad-spectrum immunosuppressive activity. In the context of
exacerbated inflammation as we described in SARS-CoV-2, they may be of benefit. Thus, clinicians
tend to use it in most critically ill patients. However, such treatment was already used during SARS
and MERS outbreaks, and led to delayed viral clearance and detrimental secondary outcomes, such as
a prolonged length of stay in intensive care units or bacterial/fungal infections [309,310]. Similar data
have been emphasized for SARS-CoV-2 in moderate disease with no clear benefit of corticosteroids
use, more adverse outcomes and delayed viral clearance [311–313]. However, benefits for a precise
corticosteroids therapy in severe COVID-19 patients either pulse corticosteroids or dexamethasone
seems beneficial [314–316]. Therefore, such therapy could be of use rather at a late stage of ARDS.
The uncontrolled cytokine release in severe patients has been a major concern of therapeutic
trials. Anti-cytokine therapy has been used in this condition (anti-IL-6, anti-TNF-α and anti-IL-1) and
numerous clinical trials are still ongoing but a few showed promising results [317,318].
IL-6 is one of the most reported cytokines in COVID-19 pathology, notably in severe patients
exhibiting higher serum levels than mild cases. Thus, targeting its signaling is thought to be a therapy
of choice but at this time variable results are reported and there is no recommendation to use it in
routine owing to limited efficacy evidence [304]. To answer this question a phase II trial has been
approved with 330 patients using tocilizumab, an anti-human IL-6 receptor blocking monoclonal
antibody [319]. Of note, it has been reported that the tocilizumab effect does not compromise at
the same time the antiviral immunity in COVID-19 patients [320].
Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression is also known to be
upregulated and participate to hyperinflammation in patients suffering from COVID-19 [146,214,219].
Mavrilimumab is a human monoclonal antibody used in rheumatoid arthritis, which binds GM-CSF
receptor α and disrupts its downstream signaling, thus blocking GM-CSF inflammatory activity [321].
This monoclonal antibody showed promising preliminary data, when used in addition to standard
management [322]. Patients treated with mavrilimumab presented greater and faster improvement
than the control group [322]. However, these data need to be reproduced and confirmed on a larger
cohort and multicentric, double-blind, randomized, placebo-controlled studies.
COVID-19 patients exert a delayed IFN type I response leading to an exacerbated secondary
cytokine response, responsible for disease severity. Supporting this, type I IFN could be an interesting
treatment. Lokugamage et al. showed that in vitro SARS-CoV-2 is more sensitive to type I IFN than
SARS-CoV. Regarding these results, they suggest IFN administration as a potential treatment [323,324].
IL-1 is a major actor of uncontrolled cytokine release during severe COVID-19 [318]. Anti-IL-1
therapy by anakinra was shown to be beneficial during severe sepsis [325]. During SARS-CoV-2,
anakinra treatment improved the clinical outcome and reduced mortality [304,326].
Complement is the first line of defense during infection. As aforementioned, C5a is responsible for
PMN recruitment, and thus probably participates to pulmonary injury during SARS-CoV-2 infection.
Eculizumab is a monoclonal antibody that binds C5, hence inhibiting C5a and C5b release, as well
as the membrane attack complex formation. This monoclonal antibody is predicted to be a potential
therapeutic approach [327], according to what has been explored for MERS-CoV in animal models [157].
Clathrin-mediated endocytosis is a possible SARS-CoV-2 way of cell entry and is regulated by
members of the numb-associated kinase (NAK) family [325]. Janus kinase (JAK) inhibitors, notably
baracitinib used in rheumatoid arthritis, have been evoked as potential therapeutics during COVID-19
through inhibition of the NAK family [325,328]. Their anti-inflammatory effect may be beneficial in
reducing cytokine release in severe patients or detrimental by delaying the interferon response [318,325].
Results of case report and small cohort studies seem encouraging with an improvement of moderate to
severe cases and need to be pursued [329–331].
Int. J. Mol. Sci. 2020, 21, 5932 23 of 40

5.3. Stem Cell-Based Therapy

Mesenchymal stem cells (MSC) are known to have immunoregulatory activities and are for this
purpose used as an injected therapy in immune-mediated diseases. Based on these immunoregulatory
activities and the supposed pathogenesis of COVID-19, MSC or MSC-secretome injection have been
envisaged as treatment by several teams [332–336]. Of note, unlike antibody-based therapy, MSC can
target at the same time different cytokines, notably due to their diverse secretome, making these cells
a therapy of choice [332]. Thus, it appears that injection of ACE2–MSCs improved the outcomes of
seven enrolled COVID-19 patients, mainly through their immunomodulating function [333], leading
to inflammation markers reduction [336]. Nonetheless, these experiments were conducted on a very
limited number of patients and the statement established should be reconfirmed on a larger cohort.

Author Contributions: Conceptualization, G.L. and D.V.; writing—original draft preparation, G.L. and D.V.;
writing—review and editing, G.L., D.V., É.F., F.A.-P., X.G., E.N., L.R. and P.G. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Figures were created with Biorender.com.
Conflicts of Interest: The authors declare no conflict of interest.

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